Project Summary The emerging pathogen Mycobacterium abscessus and its subspecies are very closely related (referred to hereafter as M. abscessus) and cause the majority of mycobacterial infections in the USA. When inhaled, M. abscessus can cause a serious pulmonary disease in cystic fibrosis patients, immunocompromised individuals and others with underlying lung disorders. Modes of transmission are broader than once thought, as most individuals with cystic fibrosis appear to contract M. abscessus pulmonary infections via long-lived aerosols or fomites. A significant number of M. abscessus infections are also acquired in the hospital setting after surgery or upon exposure to contaminated syringes or needles (e.g. from piercings or tattoos). M. abscessus is also a leading cause of skin and soft tissue infections, especially after plastic surgery. Nosocomial infections and suspected infections from fomites are also hard to prevent because M. abscessus is resistant to most disinfectants and biocides. Finally, infections are difficult to treat because M. abscessus is one of the most antibiotic-resistant, rapidly growing nontuberculous mycobacteria. Fundamental mechanisms of M. abscessus pathogenesis and virulence are still unclear, and it is not understood why it is so extraordinarily refractory to antimicrobial drugs. Consequently, NIH is seeking applications (NOT-AI-17-016 Notice of NIAID?s Interest in Biomedical Research in non-AIDS associated, Pulmonary Non-Tuberculous Mycobacterial (NTM) Infections) to begin to fill these profound gaps. In this proposal, we hypothesize that the toxins derived from toxin-antitoxin systems present in the genomes of over 100 M. abscessus clinical isolates act by arresting cell growth and initiating a state of persistence that enables this pathogen to evade killing by antimicrobial drugs. To test this hypothesis, the goals of this R21 proposal are to apply two powerful genome-scale methods to identify which RNAs are targeted by these M. abscessus toxins and how these toxins reshape both the M. abscessus transcriptome and proteome. In addition to illuminating the mechanism of action of these toxins, we will also be able to determine if there is correlation a between the presence of one or more toxins and an increase in virulence and/or antibiotic resistance because patterns of drug resistance are known to differ among M. abscessus subsp. Overall, these studies will uncover new insights into the full spectrum of molecular events that underlie the growth regulating properties of these toxins, result in the development of useful biomarkers, and serve as a foundation for the development of more effective treatments for M. abscessus infections. !